Project Summary Millions of Americans suffer from chronic sleep disorders, which often result in other co-morbid conditions such as depression and obesity. Understanding the molecular mechanism of sleep will help us treat sleep disorders. Sleep researchers have found that 20 of the 70 human genes that encode neuropeptides have been implicated in sleep regulation. Yet little is known about how these neuropeptides interact with neural circuits to regulate sleep. I hypothesize that sleep results from the collective action of multiple neuropeptides. Neuropeptides are genetically encoded neuromodulators that can integrate environmental stimuli such as hunger and then coordinately regulate behavior by exciting or inhibiting target neurons to induce behavioral states. The evolutionary importance of neuropeptides is demonstrated by the high level of conservation of many neuropeptides from humans to worms. Genes encoding neuropeptides have been shown to play a key regulatory role in the modulation of sleep, and my proposal will determine how these neuropeptides act on a simple aversive neural circuit during sleep. I will approach these complicated questions about sleep using the genetically tractable and simple nervous system (302 neurons) of C. elegans. I will take advantage of the well-studied nervous system of C. elegans and evaluate how the sensory system is modulated by neuropeptides during sleep. There are 122 neuropeptide encoding genes in C. elegans, and my previous work found a number of specific neuropeptides that regulate sleep in C. elegans. This investigation will elucidate the function of two classes of neuropeptides: RFamides, recently shown to regulate sleep across metazoan phylums, and Tachykinin, a neuropeptide implicated in vertebrate sleep. My project will apply the next generation of neurobiology techniques, such as optogenetics and multi-neuron imaging, to control and record the activity of neural circuits during sleep. Optogenetics uses light-activated channels and pumps to activate and inhibit individual neurons. Multi-neuron imaging techniques for visualization of nervous systems activity in vivo, has provided neuroscientists a window from which they can visualize the activity of a nervous system. I plan to combine the study of neuromodulation with optogenetics and multi-neuron imaging in the context of sleep. My previous experiments suggest that sleep results from the collective action of multiple neuropeptides. I hypothesize that these neuropeptides act at distinct cellular sites. I hope to provide the first complete model that describes how neuromodulators regulate an aversive neural circuit during sleep, which will have major evolutionary implications for the neuropeptidergic regulation of sensory depression in vertebrates.